Lorenzen A, Engelhardt J, Kerst B, Schwabe U
Universitat Heidelberg, Pharmakologisches Institut, Germany.
Biochem Pharmacol. 1998 Feb 15;55(4):455-64. doi: 10.1016/s0006-2952(97)00483-8.
The localization of the low-affinity adenosine binding protein adenotin-1 with respect to distribution in rat organs and subcellular compartments was investigated. Adenotin-1 was characterized by 5'-N-ethylcarboxamido[2,8-3H]adenosine ([3H]NECA) binding and Western blotting. Cytosolic as well as membrane fractions of all tissues contained adenotin-1. Highest levels of membrane-bound adenotin-1 were found in the liver (liver > kidney approximately spleen approximately lung > forebrain approximately cerebellum > fat heart - striated muscle), whereas highest levels of cytosolic adenotin-1 were detected in spleen, liver, lung and fat. Subcellular fractions from rat liver were prepared by differential and density gradient centrifugation. Like the homologous proteins endoplasmin or gp96, adenotin-1 is enriched in the endoplasmic reticulum. Cytosolic and membrane-bound adenotin-1 species are pharmacologically distinct, because in the liver particulate fraction adenotin-1 showed a more rapid binding kinetics, a twofold lower affinity for [3H]NECA (KD 227 nM vs. 105 nM) and a sevenfold higher affinity for 2-chloroadenosine than the cytosolic protein (Ki 1.48 microM vs. 9.25 microM). In rat liver cytosol, two different binding sites were found, which differed in [3H]NECA binding kinetics and displayed a hundredfold difference in their affinity for 2-chloro-5'-N-methylcarboxamidoadenosine (Ki 45.8 nM vs. 4.76 microM). The presence of adenotin-1 in subcellular fractions, as determined by radioligand binding, was confirmed by Western blotting. Adenotin-1 was detected as a 98-kDa band in all rat liver subcellular fractions, which agrees with the molecular mass determined for the purified protein. In the cytosol, a 65-kDa hand was labeled more intensely than the 98-kDa band. This additional band probably represents the pharmacologically distinct species of adenotin-1 found in the cytosol.
研究了低亲和力腺苷结合蛋白腺嘌呤素-1在大鼠器官和亚细胞区室中的分布定位。通过5'-N-乙基羧酰胺基[2,8-³H]腺苷（[³H]NECA）结合和蛋白质印迹法对腺嘌呤素-1进行了表征。所有组织的胞质和膜部分均含有腺嘌呤素-1。膜结合腺嘌呤素-1的最高水平见于肝脏（肝脏＞肾脏≈脾脏≈肺＞前脑≈小脑＞脂肪心脏-横纹肌），而胞质腺嘌呤素-1的最高水平则在脾脏、肝脏、肺和脂肪中检测到。通过差速离心和密度梯度离心制备大鼠肝脏的亚细胞组分。与同源蛋白内质素或gp96一样，腺嘌呤素-1在内质网中富集。胞质和膜结合的腺嘌呤素-1种类在药理学上有所不同，因为在肝脏微粒体部分，腺嘌呤素-1表现出更快的结合动力学，对[³H]NECA的亲和力低两倍（KD 227 nM对105 nM），对2-氯腺苷的亲和力比胞质蛋白高七倍（Ki 1.48 μM对9.25 μM）。在大鼠肝脏胞质溶胶中，发现了两个不同的结合位点，它们在[³H]NECA结合动力学上有所不同，对2-氯-5'-N-甲基羧酰胺基腺苷的亲和力相差百倍（Ki 45.8 nM对4.76 μM）。通过放射性配体结合测定的亚细胞组分中腺嘌呤素-1的存在，经蛋白质印迹法得到证实。在所有大鼠肝脏亚细胞组分中，腺嘌呤素-1被检测为一条98 kDa的条带，这与纯化蛋白测定的分子量一致。在胞质溶胶中，一条65 kDa的条带比98 kDa的条带标记更强。这条额外的条带可能代表在胞质溶胶中发现的药理学上不同的腺嘌呤素-1种类。
